Leghaemoglobin Content Research Articles

Effect of co-inoculation with Bradyrhizobium japonicum and Azospirillum brasilense on soybean plants

In order to assess the effects of co-inoculation with both Bradyrhizobium japonicum and Azospirillum brasilense on soybean plants, a pot experiment under growth controlled conditions was carried out. Four-week-old plants were harvested and analyzed for shoot, root and nodule dry weight, nitrogen content, nodule number and distribution, nodule protein and leghaemoglobin content, and nitrogenase and glutamine synthetase activities. Nodule number and nodule dry weight were not affected by dual inoculation; however, co-inoculated plants showed a significantly higher proportion of nodules attached to the main root and located in the upper 3 cm of the root system. Although no significant difference in total dry matter production could be detected, nitrogen content of dual inoculated plants was significantly increased (23 % over B. japonicum-single. inoculated plants). Accordingly, a strong stimulation of acetylene reduction activity was observed for this treatment. Likewise, leghaemoglobin content significantly increased (39 %). These results suggest that co-inoculation with B. japonicum and A. brasilense leads to an increased number of the most active nodules, therefore, to a greater nitrogen fixation and assimilation. In addition, an enhancement of nodule effectiveness due to A. brasilense presence in the root system is suggested.

Nodule structure and functioning in Cicer arietinum as affected by nitrate

Chickpea (Cicer arietinum L.) cv. C-235 inoculated with Rhizobium sp. (Cicer) strain cv4Az was raised in sand culture under natural conditions with nitrogen-free nutrient solution. 45-d-old plants were treated with 20 and 50 mM KNO3 and sampling made 2 and 6 d after treatment. KNO3 application induced premature nodule senescence. Light microscopic investigations showed that KNO3 treatments resulted in structural degradation of the central bacteroidal tissue. The mass of green nodules increased by 35 % under these treatments. This was accompanied by a rapid decline in leghemoglobin (Lb) content of the nodules being 51 - 67 % lower than in control. The total soluble nodule proteins showed relatively minor changes under KNO3 treatments thus suggesting preferential degradation of Lb. These changes were associated with a rapid decline in N2-fixing activity. However, the decline in total soluble sugars was relatively minor as compared to acetylene reducing activity, thus indicating that sugar deprivation is not the cause of decreased nitrogen fixation ability. Glutathione reductase and ascorbate peroxidase activity showed a 10 - 20 % decrease in comparison with the control. Accumulation of H2O2 and structural degradation of the nodular tissue are considered to be the factors leading to nodule senescence under nitrate treatments.

Effect of salt stress on antioxidant defence system in soybean root nodules

The antioxidant defence systems of soybean (Glycine max (L.) Merr) nodules responded differently to 50 and 200 mM NaCl. At 50 mM NaCl, leghaemoglobin content and nitrogenase activity remained unaltered but there was an overall increase in the antioxidant enzymes (ascorbate peroxidase, catalase, glutathione reductase and superoxide dismutase) and in reduced glutathione. After returning the salinised nodules to a non-saline environment (recovery), the enzymatic activities returned to the initial values but reduced glutathione remained high with respect to the controls measured at the end of the experiment (final controls). Severe salt treatment reduced the leghaemoglobin content and nitrogenase activity by 31% and 50%, respectively. Ascorbate peroxidase, catalase and glutathione reductase activities decreased between 30 and 100% while superoxide dismutase and reduced glutathione increased over the controls by 19% and 30% respectively. After recovery, glutathione reductase increased over the final controls and reduced glutathione remained as under 50 mM NaCl. Malondialdehyde content and total protein remained unchanged in nodules treated with the two salt concentrations. These results suggest that under mild saline stress, the elevated levels of the antioxidant enzymes and reduced glutathione protect nodules against the activated oxygen species thus avoiding lipid and protein peroxidation, and leghaemoglobin breakdown. However, severe saline treatment produced an irreversible decay in the leghaemoglobin content and nitrogenase activity despite the high reduced glutathione level and glutathione reductase activity.

Alleviation of water stress effects in cowpea by Bradyrhizobium spp. inoculation

Experiments were carried out to investigate the effects of different degrees of water stress on cowpea in the presence and absence of Bradyrhizobium spp. inoculation and to evaluate physiological responses to stress. The soil used was Yellow Latosol, pH 6.3 and the crop used was cowpea (Vigna unguiculata (L.) Walp.) cv. ‘IPA 204’. Stress was applied continuously by the control of matric potential (ψ m ) through a porous cup. The lowered soil ψ m had a direct effect on the N2 fixation, but the strains Bradyrhizobium introduced by inoculation in the cowpea plants were superior to the indigenous strain demonstrating the importance of inoculation in the stressed plants. At the more negative ψ m plants inoculated with the strains EI 6 formed associations of greater symbiotic efficiency which helped the cowpea plants to withstand drought stress better than the strain BR 2001 and the uninoculated control. The leghaemoglobin concentration was not inhibited in the drought-stressed plants at ψ m -70 kPa when inoculated with the strain EI 6, which confered a differential degree of drought resistance in plants. The ψ w declined in the stressed plants reaching values of -1.0 MPa which was sufficient to cause disturbance in nodulation and biomass production.

Effects of long‐term nitrogen dioxide fumigation and nitrate supply on nitrite and Leghemoglobin concentrations, pH, and nitrogenase activity of soybean root nodules

Exposing 12‐day‐old soybean plants to 0.2 ppm nitrogen dioxide (NO2) for four weeks increased the nitrite concentration and acidity, and decreased the Leghemoglobin (LHb) concentration and the nitrogenase activity of root nodules. The supply of 1 mol.m‐3 nitrate to the roots intensified the nitrite accumulation, decreased the acidity of the nodules, and alleviated the inhibition of nitrogenase activity by NO2 fumigation. These results suggested that the inhibition of nitrogen (N2) fixation by N fertilizer supply might relate to the acid‐alkali balance in nodules.

Efficiency of the nodulating Rhizobium leguminosarum bv. viciae population under the influence of slurry deposition and slope position

In a three year study (1993, 1994 and 1995) the effect of slurry deposition, landscape position and slope exposure in a grass-covered arid region on the leghemoglobin content (Lb) of pea nodules was studied. Until 1990 this region was used for the deposition of bovine slurry over a period of five years. The leghemoglobin content of legume nodules is, at different stages of plant development, correlated with the activity of nitrogenase measured by acetylene reduction assay. The total leghemoglobin concentration (Lb/mg nodule) was used to measure the physiological activity of the nodulating Rhizobium leguminosarum bv. viciae population. A simple spectroscopic method was adapted to determine the content of leghemoglobin in the nodules of two varieties of Pisum sativum. The effect of slurry on the nodule leghemoglobin content was more pronounced at the northern exposed slope, where significant different leghemoglobin contents at nonpolluted and polluted sites could be determined in the first two years of sampling. At the southern slope no effect of slurry on the leghemoglobin content of the nodulating population was found, whereas significant differences were determined between slope position and year of sampling. The leghemoglobin content/mg nodule was higher at the southern slope in all three years compared to the northern slope and in 1995 the nodules from all sites had a significant higher leghemoglobin content compared to 1993 and 1994. The relative differences between the individual positions, measured in one year, were reproducible in every year.

Natural and dark-induced nodule senescence in chickpea: nodule functioning and H<sub>2</sub>O<sub>2</sub> scavenging enzymes

An investigation was carried out on chickpea (Cicer arietinum L.) cv. C-235 inoculated withRhizobium sp.Cicer strain cv 4 Azr. Nodule functioning was monitored at 15 d intervals starting from 45 days after sowing (DAS) and inoculation in order to study nodule development and senescence under natural and stress conditions (dark treatments of 18 and 66 h). Maximum rate of N2-fixation was observed between 50 - 60 DAS. After this acetylene reducing activity (ARA) fell and it was negligible 75 DAS. This decline in ARA with ageing of plants and nodules was accompanied by a decline in leghemoglobin content and greening of the nodules. When 60 % of the nodule tissue had turned green 75 DAS, a sharp increase in nodule peroxidase activity (3.7 fold) was observed whereas the catalase activity was reduced by 50 % in comparison with the control. The glutathione-reductase and ascorbate-peroxidase activity followed a trend parallel to that in N2-fixation, but the variation was much smaller. The changes in the total soluble carbohydrates, cytosolic proteins and nitrogen content per se were not expressive. Dark treatments induced premature senescence of the nodules as was evident from the marked decrease in ARA. However, the decline in leghemoglobin content was relatively small as compared to ARA. The changes in cytosolic proteins, total soluble carbohydrates, peroxidase activity, catalase activity, glutathione reductase activity and ascorbate peroxidase activity of nodules under dark-induced nodule senescence were almost parallel to those observed under natural senescence.

Water relations and nitrogen fixation in potassium fedVigna radiata nodules

Drought created by withholding the irrigation at 30 and 45 d after sowing significantly decreased relative water content (RWC) and osmotic potential (ψs) ofVigna radiata (L.) Wilczek cv. MH-83-30 nodules. Potassium fed plants showed higher RWC, whereas ψs was further declined irrespective of soil moisture levels. The nitrogenase activity and leghemoglobin content of nodules markedly decreased under drought and nodules of potassium fed plants showed better recovery after rehydration. The proline content significantly increased under drought but declined upon reirrigation. Also, the C, N and K contents of nodules significantly declined under drought.

Influence of indoleacetic-acid-producing Bacillus isolates on the nodulation of Phaseolus vulgaris by Rhizobium etli under gnotobiotic conditions

Twenty-two Bacillus spp. isolates from the rhizosphere of Phaseolus vulgaris 'Contender' were identified using Biolog™, gas chromatographic fatty acid methyl ester, and 23S rDNA analyses. Some of the Bacillus isolates produced significant amounts of the phytohormone indoleacetic acid (IAA) when grown in a liquid culture medium supplemented with 100 μg L-tryptophan/L; less IAA was produced in culture medium not supplemented with L-tryptophan. Thin-layer chromatography, high-performance liquid chromatography, gas chromatography – mass spectrometry, and the avena coleoptile bioassay were used to identify and quantify IAA produced by Bacillus isolates. Significant differences were observed in the amounts of IAA produced by different strains of Bacillus, with amounts varying from 0.40 to 4.88 μg/mL. α-Methyltryptophan-resistant mutants of Bacillus exhibited altered IAA production and excreted tryptophan into the growing medium. The IAA-producing Bacillus isolates promoted root growth and (or) nodulation when coinoculated with Rhizobium etli (TAL 182) on Phaseolus vulgaris 'Contender' under gnotobiotic conditions in growth chambers. Coinoculation resulted in increased nodule number, nodule fresh weight, nitrogenase activity, leghemoglobin content, and total soluble protein content in the root nodules of Phaseolus vulgaris. In contrast, coinoculation with α-methyltryptophan mutants resulted in decreased nodulation, indicating that Bacillus isolates have a direct effect on either the Rhizobium or the plant and the effect may not be singularly attributed to their ability to produce IAA in vitro.Key words: Bacillus, indoleacetic acid production, nodulation enhancement.

Host Genetic Variation in the Early Nodulation and Dinitrogen Fixation of Soybean

Soybeans [Glycine max (L.) Merr.] grown on soils low in N can exhibit N deficiency during early plant development. Improved early N2 fixation may alleviate this problem. Our objectives were to (i) examine genetic variation in early nodulation and N2 fixation of soybean, and (ii) identify traits useful in breeding for early nodulation and N2 fixation in this crop. Eighty‐six soybean lines showed considerable genetic variation in efficiency and speed of nodulation with Bradyrhizobium japonicum strain USDA 110. Eighteen lines were then reevaluated for nodulation efficiency with the B. japonicum strains USDA 123, UMR 161, and NA 6407 for differences in nodule number 5 to 12 d after inoculation (DAI) and for differences in plant dry weight, nodule fresh weight, acetylene reduction activity (ARA), nodule enzyme activity, leghemoglobin concentration, and nodule soluble protein concentration (SOLP) 10 to 17 DAI. The 86 lines differed in percentage roots nodulated (PRN; 26–93%) and number of nodules above the root tip mark (NARTM; 0.3–3.0), and in uppermost nodule position (− 17.5 to 19.5 mm). For the 18 lines that were reevaluated, mean PRN and NARTM values were greater for USDA 123 than for USDA 110 (65.1 vs. 57.9%; 1.4 vs. 1.0, respectively). For these lines, correlation analyses showed that PRN correlated with nodule number at 5 (r = 0.69**) but not 12 DAI, while nodule number 12 DAI correlated positively with nodule fresh weight, ARA, enzyme activity, and SOLP 17 DAI. Plant dry weight, nodule fresh weight, and nodule SOLP assayed at 17 DAI are suggested as traits for use in a breeding program to improve early nodulation and N2 fixation in soybean.

Possible reasons for relative salt stress tolerance in nodules of white lupin cv. Multolupa

The effects of different NaCl concentrations on the growth and nitrogen fixation activity of white lupin (Lupinus albus [L.]) was studied over a 6 d period. Plant growth parameters, photosynthesis and shoot respiration were unaffected by NaCl concentrations up to 150 mol m -3 . However, nitrogenase activity decreased with increased NaCl concentration up to 100 mol m -3 , whilst the O 2 diffusion resistance increased with 100 mol m -3 NaCl, but showed no further change when 150 mol m -3 NaCl was applied for 6 d. Increases in NaCl concentration decreased nodular starch content while increasing sucrose content, suggesting an osmotic regulation. These changes were associated with a 77% decrease in sucrose synthase activity. The effect on the O 2 diffusion resistance was paralleled by changes in glycoprotein content of the nodules, as determined by immunogold localization and ELISA. X-ray microanalysis studies of nodules showed that, following a 6d exposure to 150 mol m -3 NaCl, Na + ions were largely excluded from the infected zone, whilst only low levels of Cl - ions penetrated into this region. Na + entry into roots and leaves was also at a low level. Leghaemoglobin content decreased with saline stress, as did superoxide dismutase ; which decreased by 36% following exposure to 100 mol m -3 salt for 6 d. These results are discussed in relation to the relative salt tolerance of the Multolupa/ISLU-16 symbiosis.

Effect of water stress on functioning and structure ofCicer arietinum L. nodules

Chickpea (Cicer arietinum L. cv. 235) plants were grown in sand culture at moisture equal to 45-50% of sand saturation capacity under greenhouse conditions. 60 d after sowing, pots were divided into four lots, leaving one as control and sand moisture content of others was brought to 25-30% (S1), 12-15% (S2) and 5-6% (S3) of sand saturation capacity, by withholding the water supply and then maintaining the required levels gravimetrically till the harvest. Relative water content of leaves and nodule water content were measured as indices of water stress. With increase in the severity and duration of water stress nitrogenase activity and nitrogen and leghemoglobin content of the nodules decreased and the ratio of leghemoglobin components I and II were changed. Nodules developed under limited water availability showed decreased branching, breakdown of the endodermis, greater compactness and decreased vacuolation of cells in the central symbiotic tissue as compared to the control.

Response of nitrogen fixation, nodule activities, and growth to potassium supply in water‐stressed broad bean

Abstract Three‐week‐old nodulated faba bean plants were subjected to two levels of water stress (0.5 and 0.25 field capacity; soil water content of 20 and 10%) for five weeks. Half of the stressed plants was treated with potassium chloride (KC1) at 10 (K1) and 150 mg (K2)/kg soil at the beginning of water deficit. Nodulation was examined and some nodule activities were assayed. Nodulation, nitrogenase activity, total nitrogen (N), and dry matter yield were significantly decreased by increasing stress but were significantly higher with the two levels of potassium (K) supply. Leghaemoglobin and protein contents of cytosol as well as nodule protease and invertase were severely depressed by drought stress. Soluble carbohydrate contents of nodules, however, was significantly increased. Protein and leghaemoglobin contents and enzyme activities were greater with K fertilization but less soluble carbohydrate was accumulated. The results indicate that K supply, particularly at the 150 mg/kg soil level, increased faba bean resistance to water stress.

A functional relationship between leghaemoglobin and nitrogenase based on novel measurements of the two proteins in legume root nodules

A combination of physiological and structural measurements made on nodulated cowpea and soybean plants cultured with roots in different pO(2) permitted the expression of data in various ways. Values of leghemoglobin concentration and nitrogenase activity from the two legumes were expressed conventionally either on a per plant or per gram nodule fresh weight basis, and where microscopy was done, on the basis of nitrogenase-containing, N(2)-fixing units (i.e. per bacteroid, per infected cell, or per gram infected tissue). In both legumes, acetylene reduction, N fixed and ureide content expressed on the basis of whole plants or per nitrogenase-containing units were very significantly correlated with values of leghaemoglobin concentrations expressed in a similar manner. The use of mathematical correlations in this study involving leghaemoglobin concentrations and various indices of N(2) fixation indicated a strong functional relationship between the two proteins in symbiotic legumes. These findings confirm previous suggestions that leghaemoglobin and the nitrogenase complex are two proteins closely associated with N(2)-fixing efficiency in legume root nodules.

Comparison of twoPisum sativum nodulation mutants with their parental cultivar

In comparison with the parental cv. Finale the ‘RisfixC’ supernodulator exhibited higher, continuously increasing nodule number and fresh mass accumulation, but substantially lower individual nodule fresh mass, leghemoglobin concentration, and specific acetylene reduction activity of nodule tissue. There were no substantial differences between Finale and ‘RisfixC’ in total acetylene reduction, nodule leghemoglobin accumulation per nodulated root, total and specific CO2 evolution from nodulated roots and gross CO2 respiratory costs of acetylene reduction. The ‘RisfixC’ also exhibited a substantially lower plant dry mass production (by 30%), but nitrogen concentration in shoots and carotenoid concentration in leaf tissue were significantly higher by 33 and 14%, and the chlorophylla+b content insignficantly higher than in the parental cultivar. In contrast, the nodulation mutant ‘Risnod29’, exhibited a somewhat higher nodule fresh mass accumulation (by 21%) and individual nodule fresh mass (by 23%), total and specific acetylene reduction (by 49 and 19%) and a somewhat more rapid plant dry mass accumulation compared with the cv. Finale.

Effects of salt stress on growth and nitrogen fixation by pea, faba-bean, common bean and soybean plants

The effects of salinity on growth, nodulation, acetylene reduction activity (ARA), nodule leghemoglobin (Lb) content and respiratory capacity of bacteroids from pea ( Pisum sativwn cv. Lincoln), faba-bean ( Vicia faba L. var. minor cv. Alborea), bean ( Phaseolus vulgaris cv. Contender) and soybean ( Glycine max L. var. Williams) were determined. The depressive effect of saline stress (50 and 100 mM NaCl) on dry weight and ARA of nodules was directly related to the salt-induced decline in dry weight and N content in shoots. The results indicate that P. savitum was the legume most severely affected by salinity, whereas G. max was the most salt-tolerant species. Saline stress was also responsible for a decrease in cytosolic protein of nodules, specifically Lb; this effect was more pronounced in pea and bean nodules than in soybean and faba-bean nodules. Bacteroids isolated from soybean and bean nodules exhibited higher respiratory activity in the absence of energy-yielding substrates than those isolated from pea and faba-bean plants. Salt added directly to the incubation mixture of bacteroids inhibited O 2 uptake of bacteroids isolated from nodules of each legume. However, salt treatment of plants decreased respiratory capacity only in pea and faba-bean bacteroids. Inhibition of ARA under moderate saline stress may be related to the drop in bacteroid respiration. Under severe stress both the reduction in Lb content and inhibition of bacteroid respiration may be involved in salt-induced inhibition of nitrogen fixation.

Nitrogen fixation and carbon metabolism by nodules and bacteroids of pea plants under sodium chloride stress

Acetylene reduction activity (ARA) and leghemoglobin (Lb) content in nodules were sigificantly reduced when pea (Pisum sativum L. cv. Lincoln) plants were subjected to 50 mM sodium chloride stress for 3 weeks. C2H2 reduction activity by bacteriods isolated from pea nodules was drastically inhibited by saline stress, and malate appeared to be a more appropriate substrate than glucose or succinate in maintaining this activity. Salt added directly to the incubation mixture of bacteriods or to the culture medium of plants inhibited O2 uptake by bacteroids. Nodule cytosolic phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and bacteriod malate dehydrogenase (MDH; EC 1.1.1.37) activities were strongly enhanced by salt stress. Under these conditions, malate concentration was depressed in bacteroids and cytosol, whereas total soluble sugar (TSS)content slightly increased in both fractions. The effect of salt stress on TSS and malate content suggests that the utilization of carbohydrate within nodules could be inhibited during salt stress. The inhibitory effect of NaCl on N2 fixation activity of bacteroids and to the decrease in bacteroid respiration. The stimulation of fermentative metabolism induced by salinity suggests some reduction in O2 availability within the nodule. Salt stress was also responsible for a decrease of the cytosolic protein content, specifically of leghemoglobin, in the nodules.

Herbicides Effects an Nodulation, Growth and Nitrogen Yield of Faba Bean Induced by Indigenous Rhizobium leguminosarum

Summary The effect of the herbicides brominal (Bromoxynil: 20% active ingredient, 3,5-dibromo-4-hydroxyben-zonitrile) and Gramoxone (Paraquat: 24% active ingredient, 1,1-dimethyl-4,4-bipyridylium dichloride) on the growth and symbiotic nitrogen fixation of Vicia faba was studied in pots. The herbicides were applied at rates of recommended field dose as well as 2 and 3 fold-field dose to unsterilized clay soil. All doses of herbicides significantly reduced the plant growth (dry matter), nodulation, total nitrogen and carbohydrate contents of shoot and roots. Protein, leghaemoglobin and carbohydrate contents of cytosol and bacteroid fractions ware significantly inhibited when plant treated with 2 and 3-fold recommended field dose. The results suggest that both herbicides act directly to the plant and indirectly on the nodule content.

The role of copper in nitrogen fixation in Lupinus luteus L.

The effect of copper nutrition on symbiotic N2 fixation in Lupinus luteus L. was studied. Copper nutrition increased the yield, total nitrogen content and dry weight of nodules. The control plants did not produce pods. Copper deficiency limited iron uptake and its translocation to the nodules. Nodules of copper-deficient plants contained less than half the leghaemoglobin concentration of copper-adequate plants and about one third the polyphenol oxidase activity, tested with catechol as a substrate.

Effect of oil pollution on soil respiration and growth of Vigna mungo (L.) Hepper

As a result of continuous aqueous oil effluent irrigation in about 25 acres of crop field, oil compounds infiltrated up to 50 cm depth of the soil. In an oil-polluted soil, respiration was always higher than the uncontaminated soil. Total hydrocarbon content of the polluted soil was quantified by infrared spectrophotometric technique. A significant correlation was observed between oil concentration (total hydrocarbons) in the upper layer of the soil (0–25 cm) and air temperature. Seed germination, percentage of seedling emergence, number of root nodules/plant, leghaemoglobin content in root nodules, total soluble sugars, total soluble proteins, free amino acids, total chlorophyll and carotenoids and nucleic acids of the leaves of Vigna mungo (L.) Hepper grown in the polluted soil decreased significantly due to persistence of hydrocarbon. Total phenolic content of the leaves in the polluted plant increased significantly.